11a, 12-DERIVATIVES OF TETRACYCLINE COMPOUNDS

ABSTRACT

11a,12-dehydrotetracycline compounds are described.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/348,608 (Allowed) filed Feb. 6, 2006, which claims the benefit ofU.S. Provisional Application No. 60/650,031 filed Feb. 4, 2005, theentire contents of each of the aforementioned applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbacteriocidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later, oxytetracycline became available. The elucidation ofthe chemical structure of these compounds confirmed their similarity andfurnished the analytical basis for the production of a third member ofthis group in 1952, tetracycline. A new family of tetracyclinecompounds, without the ring-attached methyl group present in earliertetracyclines, was prepared in 1957 and became publicly available in1967; and minocycline was in use by 1972.

Recently, research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration. New tetracycline analogues have also beeninvestigated which may prove to be equal to or more effective than theoriginally introduced tetracycline compounds. Examples include U.S. Pat.Nos. 2,980,584; 2,990,331; 3,062,717; 3,165,531; 3,454,697; 3,557,280;3,674,859; 3,957,980; 4,018,889; 4,024,272; and 4,126,680. These patentsare representative of the range of pharmaceutically active tetracyclineand tetracycline analogue compositions.

Historically, soon after their initial development and introduction, thetetracyclines were found to be highly effective pharmacologicallyagainst rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,inclusion conjunctivitis, and psittacosis. Hence, tetracyclines becameknown as “broad spectrum” antibiotics. With the subsequent establishmentof their in vitro antimicrobial activity, effectiveness in experimentalinfections, and pharmacological properties, the tetracyclines as a classrapidly became widely used for therapeutic purposes. However, thiswidespread use of tetracyclines for both major and minor illnesses anddiseases led directly to the emergence of resistance to theseantibiotics even among highly susceptible bacterial species bothcommensal and pathogenic (e.g., pneumococci and Salmonella). The rise oftetracycline-resistant organisms has resulted in a general decline inuse of tetracyclines and tetracycline analogue compositions asantibiotics of choice.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains, at least in part, to12-dehydrotetracycline compounds. In a further embodiment, the inventionpertains to tetracycline compounds of formula (I):

wherein

R¹ is hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, amido,alkylamino, amino, arylamino, alkylcarbonyl, arylcarbonyl,alkylaminocarbonyl, alkoxy, alkoxycarbonyl, alkylcarbonyloxy,alkyloxycarbonyloxy, arylcarbonyloxy, aryloxy, thiol, alkylthio,arylthio, alkenyl, heterocyclic, hydroxy, or halogen, optionally linkedto R² to form a ring;

R² is hydrogen, alkyl, halogen, alkenyl, alkynyl, aryl, hydroxyl, thiol,cyano, nitro, acyl, formyl, alkoxy, amino, alkylamino, heterocyclic, orabsent, optionally linked to R¹ to form a ring;

R^(2′), R^(2″), R^(4a), and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, and R¹² are each hydrogen, alkyl, aryl, benzyl, arylalkyl, or apro-drug moiety;

R⁴ and R^(4′) are each independently NR^(4a)R^(4b), alkyl, alkenyl,alkynyl, hydroxyl, halogen, or hydrogen;

R⁵ and R^(5′) are each independently hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR⁷)₀₋₁C(═W¹)WR^(7a);

R⁸ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(8c))₀₋₁C(=E¹)ER^(8a);

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃ (NR^(9c))₀₋₁C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a), R^(8b), R^(8c),R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c)R^(9d), R^(9e), and R^(9f)are each independently hydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,heterocyclic, heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

E is CR^(8d)R^(8e), S, NR^(8b) or O;

E′ is O, NR⁸, or S;

Q is a double bond when R^(1′) and R² are absent, Q is a single bondwhen R^(1′) and R² are each independently hydrogen, alkyl, halogen,hydroxyl, thiol, alkenyl, alkynyl, aryl, acyl, formyl, alkoxy, amino,alkylamino, or heterocyclic;

W is CR^(7d)R^(7e), S, NR^(7b) or O;

W′ is O, NR^(7f), or S;

X is CHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f), and pharmaceutically acceptable salts, esters,prodrugs, and enantiomers thereof.

In yet another embodiment, the invention pertains, at least in part, totetracycline compound of formula (II):

wherein

R¹ is hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, amido,alkylamino, amino, arylamino, alkylcarbonyl, arylcarbonyl,alkylaminocarbonyl, alkoxy, alkoxycarbonyl, alkylcarbonyloxy,alkyloxycarbonyloxy, arylcarbonyloxy, aryloxy, thiol, alkylthio,arylthio, alkenyl, heterocyclic, hydroxy, or halogen;

R^(2′), R^(2″), R^(4a), and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, and R¹² are each independently hydrogen, alkyl, aryl, benzyl,arylalkyl, or a pro-drug moiety;

R⁴ is NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R⁵ and R^(5′) are each independently hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a);

R⁸ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(8c))₀₋₁C(=E′)ER^(8a);

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(9c))₀₋₁C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a), R^(8b), R^(8c),R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c), R^(9d), R^(9e), andR^(9f) are each independently hydrogen, acyl, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

E is CR^(8d)R^(8e), S, NR^(8b) or O;

E′ is O, NR^(8f), or S;

W is CR^(7d)R^(7e), S, NR^(7b) or O;

W′ is O, NR⁷, or S;

X is CHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR⁹, and pharmaceutically acceptable salts, esters andenantiomers thereof.

The invention also includes, for example, method for treating atetracycline responsive state in a subject. The methods includeadministering to a subject an effective amount of a tetracyclinecompound of the invention (e.g., a compound of any one of formula I, IIor otherwise described herein).

The invention also pertains, at least in part, to pharmaceuticalcompositions which comprise an effective amount of a tetracyclinecompound of the invention (e.g., a tetracycline compound of formula I,II, or otherwise described herein) and a pharmaceutically acceptablecarrier.

The invention also pertains, at least in part, to a method forsynthesizing dehydrotetracycline compounds. The method includescontacting a tetracycline compound with an effective amount of areducing agent to form a 12-hydroxy tetracycline compound; andcontacting the 12-hydroxy tetracycline compound with a dehydrationagent, to form a dehydrotetracycline compound.

The invention also pertains, at least in part, to a method forsynthesizing C11a-C12 cleaved tetracycline compounds, by contacting adehydrotetracycline compound with a cleavage reagent, such that aC11a-C12 cleaved tetracycline compound is formed.

In another embodiment, the invention pertains to a method ofsynthesizing a substituted tetracycline compound, by contacting adehydrotetracycline compound with a reactive agent, such that asubstituted tetracycline compound is formed.

DETAILED DESCRIPTION OF THE INVENTION 1. Dehydrotetracycline Compounds

The invention pertains, at least in part, to novel 11a, and/or12-position derivatives of tetracyclines and methods of producing11a,12-dehydrotetracycline compounds.

The term “tetracycline compound” includes many compounds with a similarring structure to tetracycline. Examples of tetracycline compoundsinclude: tetracycline, oxytetracycline, chlortetracycline,demeclocycline, doxycycline, chelocardin, minocycline, rolitetracycline,lymecycline, sancycline, methacycline, apicycline, clomocycline,guamecycline, meglucycline, mepylcycline, penimepicycline, pipacycline,etamocycline, and penimocycline. Other derivatives and analoguescomprising a similar four ring structure are also included. The termincludes 4-dedimethylamino derivatives. Table 1 depicts tetracycline andseveral known tetracycline derivatives. The tetracycline compounds maybe unsubstituted at any position or further substituted, for example, atthe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12a or 13 position of the ring. TheC12 position on each of the tetracycline compounds shown in Table 1 isindicated by an arrow.

TABLE I

  Tetracycline

  Oxytetracycline

  Methacycline

  Doxycycline

Other tetracycline compounds which may be modified using the methods ofthe invention include, but are not limited to,6-demethyl-6-deoxy-4-dedimethylaminotetracycline; tetracyclino-pyrazole;7-chloro-4-dedimethylaminotetracycline;4-hydroxy-4-dedimethylaminotetracycline;12a-deoxy-4-dedimethylaminotetracycline;5-hydroxy-6a-deoxy-4-dedimethylaminotetracycline;4-dedimethylamino-12a-deoxyanhydrotetracycline;7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline;tetracyclinonitrile; 4-oxo-4-dedimethylaminotetracycline 4,6-hemiketal;4-oxo-11a C1-4-dedimethylaminotetracycline-4,6-hemiketal;5a,6-anhydro-4-hydrazon-4-dedimethylamino tetracycline;4-hydroxyimino-4-dedimethylamino tetracyclines;4-hydroxyimino-4-dedimethylamino 5a,6-anhydrotetracyclines;4-amino-4-dedimethylamino-5a, 6 anhydrotetracycline;4-methylamino-4-dedimethylamino tetracycline;4-hydrazono-11a-chloro-6-deoxy-6-demethyl-6-methylene-4-dedimethylaminotetracycline; tetracycline quaternary ammonium compounds;anhydrotetracycline betaines; 4-hydroxy-6-methyl pretetramides; 4-ketotetracyclines; 5-keto tetracyclines; 5a, 11a dehydro tetracyclines; 11aC1-6, 12 hemiketal tetracyclines; a C1-6-methylene tetracyclines; 6, 13diol tetracyclines; 6-benzylthiomethylene tetracyclines; 7,11a-dichloro-6-fluoro-methyl-6-deoxy tetracyclines; 6-fluoro(α)-6-demethyl-6-deoxy tetracyclines; 6-fluoro(β)-6-demethyl-6-deoxytetracyclines; 6-α acetoxy-6-demethyl tetracyclines; 6-βacetoxy-6-demethyl tetracyclines; 7,13-epithiotetracyclines;oxytetracyclines; 11a halogens of tetracyclines; 12a formyl and otheresters of tetracyclines; 5, 12a esters of tetracyclines; 10,12a-diesters of tetracyclines; 12-a-deoxyanhydro tetracyclines;6-demethyl-12a-deoxy-7-chloroanhydrotetracyclines; B-nortetracyclines;7-methoxy-6-demethyl-6-deoxytetracyclines;6-demethyl-6-deoxy-5a-epitetracyclines; 8-hydroxy-6-demethyl-6-deoxytetracyclines; monardene; chromocycline; 5a methyl-6-demethyl-6-deoxytetracyclines; 6-oxa tetracyclines, and 6 thia tetracyclines. Otherexamples of tetracycline compounds which may be used to formdehydrotetracycline compounds of the invention include those describedin U.S. Published Applications 20040002481 and 20050282787, eachincorporated herein by reference.

The term “12-dehydrotetracycline compounds” or “12-position tetracyclinederivatives” includes tetracycline compounds which contain a substituentother than a hydroxy at the C12 position and/or a substitution at the11a position. In an embodiment, the dehydrotetracycline compound isdehydrotetracycline (e.g., wherein R⁴ is NR^(4a)R^(4b); R^(4a) andR^(4b) are methyl, R^(4′), R⁵, and R^(5′) are hydrogen and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydroxy);dehydrodoxycycline (e.g., wherein R⁴ is NR^(4a)R^(4b); R^(4a) and R^(4b)are methyl, R⁵ is hydroxyl, R^(4′) and R^(5′) are hydrogen, and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydrogen);dehydrominocycline (wherein R⁴ is NR^(4a)R^(4b); R^(4a) and R^(4b) aremethyl; R^(4′), R^(5′), and R⁵ are hydrogen and X is C⁶R^(6′) wherein R⁶and R^(6′) are hydrogen atoms, and R⁷ is dimethylamino); ordehydrosancycline (wherein R⁴ is NR^(4a)R^(4b); R^(4a) and R^(4b) aremethyl; R^(4′), R^(5′), and R⁵ are hydrogen and X is CR⁶R^(6′) whereinR⁶ and R^(6′) are hydrogen atoms. In one embodiment, R⁴ and R^(4′) areeach hydrogen or the oxygen of a carbonyl group. The terms includecompounds of formula (I) and (II).

wherein

R¹ is hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, amido,alkylamino, amino, arylamino, alkylcarbonyl, arylcarbonyl,alkylaminocarbonyl, alkoxy, alkoxycarbonyl, alkylcarbonyloxy,alkyloxycarbonyloxy, arylcarbonyloxy, aryloxy, thiol, alkylthio,arylthio, alkenyl, heterocyclic, hydroxy, or halogen, optionally linkedto R² to form a ring;

R² is hydrogen, alkyl, halogen, alkenyl, alkynyl, aryl, hydroxyl, thiol,cyano, nitro, acyl, formyl, alkoxy, amino, alkylamino, heterocyclic, orabsent, optionally linked to R¹ to form a ring;

R^(2′), R^(2′), R^(4a), and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, and R¹² are each hydrogen, alkyl, aryl, benzyl, arylalkyl, or apro-drug moiety;

R⁴ and R^(4′) are each independently NR^(4a)R^(4b), alkyl, alkenyl,alkynyl, hydroxyl, halogen, or hydrogen;

R⁵ and R^(5′) are each independently hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a);

R⁸ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(8c))₀₋₁C(=E′)ER^(8a);

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(9c))₀₋₁C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a), R^(8b), R^(8c),R^(8d), R^(8e), R^(9a), R^(9b), R^(9c), R^(9d)R^(9e), and R^(9f) areeach independently hydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,heterocyclic, heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

E is CR^(8d)R^(8e), S, NR^(8b) or O;

E′ is O, NR^(8f), or S;

Q is a double bond when R^(1′) and R² are absent, Q is a single bondwhen R^(1′) and R² are each independently hydrogen, alkyl, halogen,hydroxyl, thiol, alkenyl, alkynyl, aryl, acyl, formyl, alkoxy, amino,alkylamino, or heterocyclic;

W is CR^(7d)R^(7e), S, NR^(7b) or O;

W′ is O, NR^(7f), or S;

X is CHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f), and pharmaceutically acceptable salts, esters,prodrugs, and enantiomers thereof.

In another embodiment, the invention pertains to tetracycline compoundsof formula (II):

wherein

R¹ is hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, amido,alkylamino, amino, arylamino, alkylcarbonyl, arylcarbonyl,alkylaminocarbonyl, alkoxy, alkoxycarbonyl, alkylcarbonyloxy,alkyloxycarbonyloxy, arylcarbonyloxy, aryloxy, thiol, alkylthio,arylthio, alkenyl, heterocyclic, hydroxy, or halogen;

R^(2′), R^(2′), R^(4a), and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, and R¹² are each independently hydrogen, alkyl, aryl, benzyl,arylalkyl, or a pro-drug moiety;

R⁴ is NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R⁵ and R^(5′) are each independently hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a);

R⁸ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR)₀₋₁C(=E′)ER^(8a);

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(9c))₀₋₁C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(8a), R^(8b), R^(8c),R^(8d), R^(8e), R^(8f), R^(9a), R^(9b), R^(9c), R^(9d), R^(9e), andR^(9f) are each independently hydrogen, acyl, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

E is CR^(8d)R^(8e), S, NR^(8b) or O;

E′ is O, NR⁸, or S;

W is CR^(7d)R^(7e), S, NR^(7b) or O;

W′ is O, NR^(7f), or S;

X is CHC(R¹³Y′Y), C═CR¹³Y, CR^(6′)R⁶, S, NR⁶, or O;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR⁹, and pharmaceutically acceptable salts, esters andenantiomers thereof.

In another embodiment, the tetracycline compound of formula I or II is a12-dehydro sancycline compound, wherein R^(2′), R³, R¹⁰, R¹¹, and R¹²are each hydrogen or a prodrug moiety; R⁴ is NR^(4a)R^(4b); R^(4a) andR^(4b) are each alkyl; X is CR⁶R^(6′); and R^(2″), R^(4′), R⁵, R^(5′),R⁶, and R^(6′) are each hydrogen.

In another embodiment, the tetracycline compound of formula I or II, isa 12-dehydro tetracycline compound, wherein R⁴ is NR^(4a)R^(4b); R^(4a)and R^(4b) are each alkyl; R⁵ and R^(5′) are hydrogen and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydroxy.

In another embodiment, the tetracycline compound of formula I or II is a12-dehydro doxycycline compound, wherein R⁴ is NR^(4a)R^(4b); R^(4a) andR^(4b) are each alkyl (e.g., methyl); R⁵ is hydroxyl; X is CR⁶R^(6′); R⁶is methyl; and R^(5′) and R^(6′) are hydrogen.

In another embodiment, the tetracycline compound of formula I or II is a12-dehydro minocycline compound, wherein R⁴ is NR^(4a)R^(4b); R^(4a) andR^(4b) are each alkyl (e.g., methyl); X is CR⁶R^(6′); R⁵, R^(5′), R⁶ andR^(6′) are hydrogen atoms and R⁷ is dimethylamino.

In an embodiment, the invention pertains to tetracycline compounds offormula I or II, wherein R¹ is hydrogen, halogen (e.g., fluorine,chlorine, bromine, iodine, etc.), hydroxy, thiol, amino, cyano, acyl,alkoxy, carboxyl, amido, alkyl, alkenyl, alkynyl, aryl, heterocyclic,alkylamino, or any other substituent which allows the tetracyclinecompound to perform its intended function.

In another embodiment, the invention pertains to tetracycline compoundsof formula I, wherein Q is a single bond. When Q is a single bond, theinvention pertains to tetracycline compounds wherein R² is hydrogen,halogen, cyano, alkyl, hydroxy, alkoxy, or any other substituent whichallows the compounds of the invention to perform their intendedfunction. In one particular embodiment, the invention pertains tocompounds wherein Q is a single bond, and R¹, R^(1′) and R² are eachindependently hydrogen. In another embodiment, the invention pertains totetracycline compounds of formula I, wherein Q is a double bond. Inanother embodiment, the invention pertains to tetracycline compoundswherein R¹ and R² are linked to form a ring. In one embodiment, R¹ andR² are linked to form an epoxide, a lactam, a lactone, a carboxylicring, a heterocyclic ring, or other ring structure. In one embodiment,R¹ and R² are linked to form a 3, 4, 5, 6, 7, 8, or 9 membered ring.

In a further embodiment, R⁹ is hydrogen. In another embodiment, R⁹ issubstituted or unsubstituted aryl (e.g., substituted or unsubstitutedcarbocyclic, e.g., phenyl or naphthyl; or substituted or unsubstitutedheteroaryl). R⁹ also may be substituted or unsubstituted alkenyl orsubstituted or unsubstituted alkynyl. R⁹ also may be heterocyclic oralkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl, orotherwise comprise a substituted carbonyl, acyl, acetyl, or formylmoiety.

In another further embodiment, R⁹ is substituted or unsubstituted alkyl.In a further embodiment, R⁹ is aminoalkyl, e.g., aminomethyl. In afurther embodiment, the aminoalkyl is further substituted with anysubstituent which allows the compound to perform its intended function.In a further embodiment, the aminoalkyl substituent is alkylaminomethyl.

In another embodiment, R⁹ is substituted or unsubstituted amino, e.g.,alkylamino, dialkylamino, arylamino, alkylcarbonylamino,alkylaminocarbonyl amino, arylcarbonylamino, etc. In another embodiment,R⁹ is amido. In yet another embodiment, R⁹ is cyano, halogen (e.g.,fluorine, bromine, chlorine, iodo, etc.), nitro, hydroxyl, alkoxy, orany other substituent which allows the tetracycline compound to performits intended function. In another embodiment, R⁹ is an R⁹ moietydescribed in WO 03/079984; WO 03/075857; WO 02/04406; or WO 01/74761,incorporated herein by reference in its entirety.

In a further embodiment, R⁷ is hydrogen. In another embodiment, R⁷ issubstituted or unsubstituted aryl (e.g., substituted or unsubstitutedcarbocyclic, e.g., phenyl or naphthyl; or substituted or unsubstitutedheteroaryl). R⁷ also may be substituted or unsubstituted alkenyl orsubstituted or unsubstituted alkynyl. R⁷ also may be heterocyclic oralkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl, orotherwise comprise a substituted carbonyl, acyl, acetyl, or formylmoiety.

In another further embodiment, R⁷ is substituted or unsubstituted alkyl.In a further embodiment, R⁷ is aminoalkyl, e.g., aminomethyl. In afurther embodiment, the aminoalkyl is further substituted with anysubstituent which allows the compound to perform its intended function.In a further embodiment, the aminoalkyl substituent is alkylaminomethyl.

In another embodiment, R⁷ is substituted or unsubstituted amino, e.g.,alkylamino, dialkylamino, arylamino, alkyl carbonylamino, alkylaminocarbonylamino, arylcarbonylamino, etc. In another embodiment, R⁷ isamido. In yet another embodiment, R⁷ is cyano, halogen (e.g., fluorine,bromine, chlorine, iodo, etc.), nitro, hydroxyl, alkoxy, or any othersubstituent which allows the tetracycline compound to perform itsintended function. In another embodiment, R⁷ is a 7-position moietydescribed in WO 02/04407, WO 01/74761, WO 03/079984, or WO 03/075857,incorporated herein by reference in their entirety.

In a further embodiment, R⁸ is hydrogen. In another embodiment, R⁸ issubstituted or unsubstituted aryl (e.g., substituted or unsubstitutedcarbocyclic, e.g., phenyl or naphthyl; or substituted or unsubstitutedheteroaryl). R⁸ also may be substituted or unsubstituted alkenyl orsubstituted or unsubstituted alkynyl. R⁸ also may be heterocyclic oralkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkyloxycarbonyl, orotherwise comprise a substituted carbonyl, acyl, acetyl, or formylmoiety.

In another further embodiment, R⁸ is substituted or unsubstituted alkyl.In a further embodiment, R⁸ is aminoalkyl, e.g., aminomethyl. In afurther embodiment, the aminoalkyl is further substituted with anysubstituent which allows the compound to perform its intended function.In a further embodiment, the aminoalkyl substituent is alkylaminomethyl.

In another embodiment, R⁸ is substituted or unsubstituted amino, e.g.,alkylamino, dialkylamino, arylamino, alkyl carbonylamino,alkylaminocarbonyl amino, arylcarbonylamino, etc. In another embodiment,R⁸ is amido. In yet another embodiment, R⁸ is cyano, halogen (e.g.,fluorine, bromine, chlorine, iodo, etc.), nitro, hydroxyl, alkoxy, orany other substituent which allows the tetracycline compound to performits intended function. In another embodiment, R⁸ is an R⁸ moietydescribed in WO 02/12170, WO 02/04404, or WO 03/079984, incorporatedherein by reference in their entirety.

In another embodiment, R³, R¹⁰, and R¹² are each independently hydrogen,alkyl, acyl, aryl, or arylalkyl. Other R³, R¹⁰, and R¹² moieties aredescribed in U.S. Ser. No. 10/619,653, incorporated herein by referencein its entirety. Other examples of R^(2′) and R^(2″) moieties aredescribed in U.S. Published Application 20040002481.

In one embodiment, the tetracycline compound is a 12-dehydrodoxycyclinecompound of the formula:

or a pharmaceutically acceptable salt, ester or prodrug thereof.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl(alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), andmore preferably 4 or fewer. Likewise, preferred cycloalkyls have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₁-C₆ includes alkyl groupscontaining 1 to 6 carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “arylalkyl”moiety is an alkyl substituted with an aryl (e.g.,phenylmethyl(benzyl)). The term “alkyl” also includes the side chains ofnatural and unnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxophenyl, quinoline, isoquinoline, naphthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,cycloalkenyl(alicyclic) groups (cyclopropenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substitutedcycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenylgroups. The term alkenyl further includes alkenyl groups which includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkenyl group has or fewer carbon atoms in itsbackbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain).Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in theirring structure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. It includes substituted acylmoieties. The term “substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by for example, alkyl groups,alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term “alkylamino” includes groups and compounds wherein the nitrogen is bound to atleast one additional alkyl group. The term “dialkyl amino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” or“alkyl aminoalkyl” refers to an alkyl, alkenyl, or alkynyl group boundto a nitrogen atom which is also bound to an alkyl group.

The term “amide” or “aminocarbonyl” includes compounds or moieties whichcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarbonyl” or“alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl oralkynyl groups bound to an amino group bound to a carbonyl group. Itincludes arylaminocarbonyl groups which include aryl or heteroarylmoieties bound to an amino group which is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarbonyl,”“alkenylaminocarbonyl,” “alkynylaminocarbonyl,” “arylaminocarbonyl,”“alkylcarbonylamino,” “alkenylcarbonylamino,” “alkynylcarbonylamino,”and “arylcarbonylamino” are included in term “amide.” Amides alsoinclude urea groups (aminocarbonylamino) and carbamates(oxycarbonylamino).

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom. Thecarbonyl can be further substituted with any moiety which allows thecompounds of the invention to perform its intended function. Forexample, carbonyl moieties may be substituted with alkyls, alkenyls,alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which containa carbonyl include aldehydes, ketones, carboxylic acids, amides, esters,anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl,arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “prodrug moiety” includes moieties which can be metabolized invivo and moieties which may advantageously remain esterified orotherwise protected in vivo. Preferably, the prodrugs moieties aremetabolized in vivo by esterases or by other mechanisms to hydroxylgroups or other advantageous groups. Examples of prodrugs and their usesare well known in the art (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). The prodrugs can beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form or hydroxyl with a suitable esterifying agent. Hydroxyl groupscan be converted into esters via treatment with a carboxylic acid.Examples of prodrug moieties include substituted and unsubstituted,branch or unbranched lower alkyl ester moieties, (e.g., propionoic acidesters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters(e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g.,acetyloxymethyl ester), acyloxy lower alkyl esters (e.g.,pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkylesters (e.g., benzyl ester), substituted (e.g., with methyl, halo, ormethoxy substituents) aryl and aryl-lower alkyl esters, amides,lower-alkyl amides, di-lower alkyl amides, and hydroxy amides.

It will be noted that the structure of some of the tetracyclinecompounds of this invention includes asymmetric carbon atoms. It is tobe understood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. Furthermore, thestructures and other compounds and moieties discussed in thisapplication also include all tautomers thereof.

2. Methods for Synthesizing 11a,12-Dehydrotetracycline Compounds

A method for derivatizing tetracycline compounds at the 11a and 12position's has been discovered through chemical modification viareduction of C1 keto-enolate to produce a C12 hydroxyl group. Thehydroxyl group is dehydrated to produce C11a-C12 dehydrotetracyclineswith a reactive α,β-unsaturated carbonyl functional group, as shown inScheme 1:

In one embodiment, the invention pertains to a method for synthesizing11a,12-dehydrotetracycline compounds. The method includes contacting atetracycline compound with an effective amount of a reducing agent toform a 11a,12-hydroxy tetracycline compound, and contacting the11a,12-hydroxy tetracycline compound with a dehydration agent, such thata 11a,12-dehydrotetracycline compound is formed.

The term “reducing agent” includes agents which are capable of reducingthe C12 keto-enolate to a hydroxyl group. Examples of reducing agentsare described in Comprehensive Organic Transformations (“COT”) 2^(nd)Ed., Larock, 304, 305. In one embodiment, the reducing agent is sodiumborohydride.

Dehydration agents are also known in the art. Examples of dehydrationagents are described in Comprehensive Organic Transformations (“COT”)2^(nd) Ed., Larock, 304, 305, incorporated herein by reference. In oneembodiment, the dehydration agent is an acid, e.g., trifluoroacetic acidand/or heat.

In one embodiment, the tetracycline compound is tetracycline,doxycycline, methacycline, minocycline, or sancycline. In anotherembodiment, the tetracycline compound is a tetracycline compounddescribed in, for example, WO 03/079983, WO 02/12170, WO 02/04407, WO02.04406, WO 02/04405, WO 02/04404, WO 01/74761, WO 03/079984, WO03/075857, WO 03/057169, WO 02/072545, WO 02/072506, U.S. Ser. No.10/619,653, U.S. Ser. No. 09/895,857; U.S. Ser. No. 09/895,812; U.S.Pat. No. 5,326,759; U.S. Pat. No. 5,328,902; U.S. Pat. No. 5,495,031;U.S. Pat. No. 5,495,018; U.S. Pat. No. 5,495,030; U.S. Pat. No.5,495,032; U.S. Pat. No. 5,512,553; U.S. Pat. No. 5,675,030; U.S. Pat.No. 5,843,925; U.S. Pat. No. 5,886,175; U.S. Pat. No. 6,165,999; U.S.Pat. No. 3,239,499; WO 95/22529; U.S. Pat. No. 5,064,821; U.S. Pat. No.5,589,470; U.S. Pat. No. 5,811,412, U.S. Publication No. 20040002481, orU.S. Publication No. 20050282787, each of which is hereby incorporatedby reference.

A wide range of tetracycline compounds of the invention can besynthesized using the methods of the invention. The tetracyclinecompounds of the invention can be synthesized, for example, by reactingvarious reactive agent, such as nucleophiles, with thedehydrotetracycline to produce tetracycline compounds of the invention.Examples of some of the substituted tetracyclines which can besynthesized using the methods of the invention include compounds withC12-carbon-carbon, C12-position, as shown in Scheme 2.

In Scheme 2, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), and R^(1g) are eachindependently hydrogen, alkyl, heterocyclic, aryl, alkenyl, alkynyl,alkoxy, carbonyl, acyl, halogen, cyano, amino, amido, nitro, or anyother substituent described herein which would allow the tetracyclinecompounds of the invention to perform their intended functions.

Tetracycline compounds of the invention can be synthesized using methodsand reactive agents known the art to react with α,β-unsaturated ketones.For example, anions, carbanions and alkali metals are reactive agentswhich react to give tetracycline compounds of the invention with variousR¹ substituents (Scheme 3). Other reactive agents which can be used tosynthesize the compounds of the invention with various R¹ substituentsinclude alkali metal acetylides, lithium dialkyl cuprates, lithiumdiarylcuprates with or without complexing ligands. Tetracyclinecompounds with R¹ substituents can also be formed using reactive agentssuch as anions, e.g., anions generated from ketones, aldehydes and thelike. Reactive agents also include organometallic reagents such asGrignards and organolithium and ylide reagents (COT, 351-401, Scheme 3).

Other reactive agents which can be used include those used in enol-ester(COT, 1485-1487) and enol silane (COT, 1488-1505) reactions. Otherreactive agents include hydration agents. Hydration can be used tointroduce R¹ hydroxyl groups (COT, 991-995). Tetracycline compounds ofthe invention wherein R¹ is hydroxyl can be formed also by hydroborationfollowed by oxidation to an alcohol (COT, 1005-1008). The tetracyclinecompounds having a substituted carbonyl at the R¹ position can besynthesized by carbonylation (COT, 1009-1011, 1690-1693).

Tetracycline compounds of the invention having alkyl and othersubstituents at the R¹ position can be synthesized using a variety ofreactive agents via free radical, Michael and Michael-like additionreactions, and organoboron reactions. Other possible reactive agentsinclude catalysts which can be used to synthesize various tetracyclinecompounds of the invention include aluminum, gallium, thallium, silicon,germanium, tin, lead, stilbine, titanium, zirconium, manganese, iron,cobalt, rhodium, nickel, palladium, copper, silver, zinc, mercury, andothers (COT, 1560-1616). Tetracycline compounds of the invention whereinR¹ is cyano can also be formed using reactive agents (COT, 1705-1706).Tetracycline compounds of the invention wherein R¹ is an ester can besynthesized, for example, using ester additions (COT, 1724-1725).Tetracycline compounds of the invention wherein R¹ comprises a nitrilegroup can be synthesized using nitrile addition reagents (COT,1800-1801) as the reactive agent. Tetracycline compounds wherein R¹ is aformyl or other carbonyl group can be synthesized using hydroformylationreagents (COT, 1363-1380) as the reactive agent.

Tetracycline compounds of the invention wherein R¹ is an amide can besynthesized via amide conjugations (COT, 1701).

Tetracycline compounds of the invention include compounds wherein R¹ isan primary, secondary, or tertiary amine. Examples of reactive agentsinclude primary and secondary amines. Amines can be formed usingtechniques known in the art (COT, 761-778). Tetracycline compounds ofthe invention with an amino R¹ substituent may also be used to formprodrugs based upon their reactivity with prodrug-forming reactiveagents.

Tetracycline compounds of formula I, wherein Q is a double bond and R¹is alkyl, alkenyl, alkynyl, or aryl can be synthesized using, forexample, conjugate addition reactive agents as shown in Scheme 5 (COT,1806-1841).

Tetracycline compounds of the invention wherein R² is alkyl, alkenyl,alkynyl, aryl, etc. can be synthesized, for example, using a variety ofreactive agents such as metal anionic reagents (Scheme 6) and by thealkylation of enones (COT, 1546-1557).

Tetracycline compounds wherein R² is hydroxy or a carbonyl group may besynthesized using asymmetric hydroboration (COT, 1008).

Tetracycline compounds of the invention may also be formed usingreactions such as reductions via catalytic hydrogenation, selectivehydrogenation, enantioselective hydrogenation,hydroboration-protonolysis, conjugate reduction, reduction dimerization(COT 2^(nd) Ed., Larock, pg 7-29), and coupling reactions (COT, 81).

Other tetracycline compounds of the invention wherein Q is a singlebond, can be synthesized using a variety of reactive agents such asorganosilicon reagents (COT, 108), organozirconium reagents (COT, 114),organonickel reagents (COT, 116-117), and organomercury (COT, 124)reagents.

Tetracycline compounds of the invention wherein R¹ and R² are halogenscan be formed by, for example, halogen addition reactions (COT,629-647). Tetracycline compounds of the invention wherein R¹ is alkyland R² is halogen can be formed, for example, by haloalkylationreactions (COT, 647-653). Tetracycline compounds of the inventionwherein R¹ and R² are hydroxyl can be synthesized by, for example,cis-hydroxylation (COT, 996-1001) and transhydroxylation (COT,1001-1003).

Tetracycline compounds of the invention wherein R¹ and R² arecarboxylate, alkyloxycarbonyl, aryloxycarbonyl, acetyl, hydroxyl, etc.can be synthesized using, for example, solvomercuration anddemercuration (COT, 1629-1632) reactions (Scheme 7).

In a further embodiment, the invention pertains to methods forsynthesizing C11a-C12 cleaved tetracycline compounds, by contacting adehydrotetracycline compound with a cleavage reagent, such that C11a-C12cleaved tetracycline compounds are formed. Examples of C11a-C12 cleavedtetracycline compounds include compounds of formula II. Cleavage andozonolysis reagents are described in COT 1210-1215, and 1630-1634.

Tetracycline compounds of the invention wherein R¹ and R² are linked toform a ring can be synthesized by reacting the alkene to form aheterocycle such as a lactone or lactam (COT, 1876-1904), as shown inScheme 8. Tetracycline compounds of the invention wherein R¹ and R² arelinked to form an epoxide are also included (COT 915-927).

Tetracycline compounds of the invention also include compounds whereinR¹ and R² are linked to form rings with 3 (COT, 135) or more (COT,136-186) members. Tetracycline compounds of the invention wherein R¹ andR² are linked to form a ring can also be synthesized via Diels-Alderreactions (COT 537), as well as other cyclization, reactions withketenes (COT, 1340-1345), annulations (COT, 1345-1362) and relatedreactions (COT, 537-560). Other ring forming reactions which can be usedto synthesize compounds of the invention include alkene additions usinghv or boron reagents forming oxetanes and higher member rings (COT,913-914, Scheme 6).

The tetracycline compounds of the invention also include tetracyclinecompounds with substituents at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.positions. For example in Scheme 10, a 11a,12-dehydro doxycycline issubstituted at the R⁹ position with an amino functional group. Thiscompound retains activity in inflammation models. Scheme 10 shows thederivatization of a 11a,12-dehydrotetracycline compound at the 9position.

Tetracycline compounds of the invention also include compounds in whichthe non-aromatic double bond (e.g., Q) is contacted with a hydrogenatingagent (Scheme 11). Suitable hydrogenating agents include, for example,hydrogen in combination with an appropriate catalyst, borohydridereagents, an aluminum hydride reagents and hydride salts (COT, 5-17).

Examples of reactions which can be used, in part, to synthesize portionsof the dehydrotetracycline compounds of the invention include thosedescribed in, for example, WO 03/079983, WO 02/12170, WO 02/04407, WO02.04406, WO 02/04405, WO 02/04404, WO 01/74761, WO 03/079984, WO03/075857, WO 03/057169, WO 02/072545, WO 02/072506, U.S. Ser. No.10/619,653, U.S. Ser. No. 09/895,857; U.S. Ser. No. 09/895,812; U.S.Pat. No. 5,326,759; U.S. Pat. No. 5,328,902; U.S. Pat. No. 5,495,031;U.S. Pat. No. 5,495,018; U.S. Pat. No. 5,495,030; U.S. Pat. No.5,495,032; U.S. Pat. No. 5,512,553; U.S. Pat. No. 5,675,030; U.S. Pat.No. 5,843,925; U.S. Pat. No. 5,886,175; U.S. Pat. No. 6,165,999; U.S.Pat. No. 3,239,499; WO 95/22529; U.S. Pat. No. 5,064,821; U.S. Pat. No.5,589,470; U.S. Publication No. 2005/0282787; and U.S. Pat. No.5,811,412, all incorporated herein by reference. In an embodiment, thepresent invention also pertains to 11a,12-dehydro derivatives of each ofthe tetracycline compounds disclosed in each of the above references.

In another embodiment, the invention pertains to tetracycline compoundssynthesized by the methods described herein.

3. Methods for Using Dehydrotetracycline Compounds

The invention also pertains to methods for treating a tetracyclineresponsive states in subjects, by administering to a subject aneffective amount of a tetracycline compound of the invention (e.g., acompound of Formula I, II, or otherwise described herein), such that thetetracycline responsive state is treated.

The term “treating” includes curing as well as ameliorating at least onesymptom of the state, disease or disorder, e.g., the tetracyclinecompound responsive state.

The language “tetracycline compound responsive state” or “tetracyclineresponsive state” includes states which can be treated, prevented, orotherwise ameliorated by the administration of a tetracycline compoundof the invention, e.g., a tetracycline compound of formula I, II orotherwise described herein). Tetracycline compound responsive statesinclude bacterial, viral, parasitic, and fungal infections (includingthose which are resistant to other tetracycline compounds), cancer(e.g., prostate, breast, colon, lung melanoma and lymph cancers andother disorders characterized by unwanted cellular proliferation,including, but not limited to, those described in U.S. Pat. No.6,100,248), arthritis, osteoporosis, diabetes, and other states forwhich tetracycline compounds have been found to be active (see, forexample, U.S. Pat. Nos. 5,789,395; 5,834,450; 6,277,061 and 5,532,227,each of which is expressly incorporated herein by reference). Compoundsof the invention can be used to prevent or control important mammalianand veterinary diseases such as diarrhea, urinary tract infections,infections of skin and skin structure, ear, nose and throat infections,wound infection, mastitis and the like. In addition, methods fortreating neoplasms using tetracycline compounds of the invention arealso included (van der Bozert et al., Cancer Res., 48:6686-6690 (1988)).In a further embodiment, the tetracycline responsive state is not abacterial infection. In another embodiment, the tetracycline compoundsof the invention are essentially non-antibacterial. For example,non-antibacterial tetracycline compounds of the invention may have MICvalues greater than about 4 μg/ml (as measured by assays known in theart and/or the assay given in Example 2). The tetracycline responsivestate also may be one treatable by the administration of antioxidants.

Tetracycline compound responsive states also include inflammatoryprocess associated states (IPAS). The term “inflammatory processassociated state” includes states in which inflammation or inflammatoryfactors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF,interleukins, plasma proteins, cellular defense systems, cytokines,lipid metabolites, proteases, toxic radicals, adhesion molecules, etc.)are involved or are present in an area in aberrant amounts, e.g., inamounts which may be advantageous to alter, e.g., to benefit thesubject. The inflammatory process is the response of living tissue todamage. The cause of inflammation may be due to physical damage,chemical substances, micro-organisms, tissue necrosis, cancer or otheragents. Acute inflammation is short-lasting, lasting only a few days. Ifit is longer lasting however, then it may be referred to as chronicinflammation.

IPAF's include inflammatory disorders. Inflammatory disorders aregenerally characterized by heat, redness, swelling, pain and loss offunction. Examples of causes of inflammatory disorders include, but arenot limited to, microbial infections (e.g., bacterial and fungalinfections), physical agents (e.g., burns, radiation, and trauma),chemical agents (e.g., toxins and caustic substances), tissue necrosisand various types of immunologic reactions.

Examples of inflammatory disorders include, but are not limited to,osteoarthritis, rheumatoid arthritis, acute and chronic infections(bacterial and fungal, including diphtheria and pertussis); acute andchronic bronchitis, acne; sinusitis, and upper respiratory infections,including the common cold; acute and chronic gastroenteritis andcolitis; acute and chronic cystitis and urethritis; acute and chronicdermatitis; acute and chronic conjunctivitis; acute and chronicserositis (pericarditis, peritonitis, synovitis, pleuritis andtendinitis); uremic pericarditis; acute and chronic cholecystis; acuteand chronic vaginitis; acute and chronic uveitis; drug reactions; insectbites; burns (thermal, chemical, and electrical); and sunburn.

Tetracycline compound responsive states also include NO associatedstates. The term “NO associated state” includes states which involve orare associated with nitric oxide (NO) or inducible nitric oxide synthase(iNOS). NO associated state includes states which are characterized byaberrant amounts of NO and/or iNOS. Preferably, the NO associated statecan be treated by administering tetracycline compounds of the invention,e.g., a compound of formula I, II, or otherwise described herein. Thedisorders, diseases and states described in U.S. Pat. Nos. 6,231,894;6,015,804; 5,919,774; and 5,789,395 are also included as NO associatedstates. The entire contents of each of these patents are herebyincorporated herein by reference.

Other examples of NO associated states include, but are not limited to,malaria, senescence, diabetes, vascular stroke, neurodegenerativedisorders (Alzheimer's disease & Huntington's disease), cardiac disease(reperfusion-associated injury following infarction), juvenile diabetes,inflammatory disorders, osteoarthritis, rheumatoid arthritis, acute,recurrent and chronic infections (bacterial, viral and fungal); acuteand chronic bronchitis, sinusitis, and respiratory infections, includingthe common cold; acute and chronic gastroenteritis and colitis; acuteand chronic cystitis and urethritis; acute and chronic dermatitis; acuteand chronic conjunctivitis; acute and chronic serositis (pericarditis,peritonitis, synovitis, pleuritis and tendonitis); uremic pericarditis;acute and chronic cholecystis; cystic fibrosis, acute and chronicvaginitis; acute and chronic uveitis; drug reactions; insect bites;burns (thermal, chemical, and electrical); and sunburn.

The term “inflammatory process associated state” also includes, in oneembodiment, matrix metalloproteinase associated states (MMPAS). MMPASinclude states characterized by aberrant amounts of MMPs or MMPactivity. These are also include as tetracycline compound responsivestates which may be treated using compounds of the invention, e.g., atetracycline compound of formula I, II, or otherwise described herein.

Examples of matrix metalloproteinase associated states (“MMPAS's”)include, but are not limited to, arteriosclerosis, corneal ulceration,emphysema, osteoarthritis, multiple sclerosis (Liedtke et al., Ann.Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol. 1997,72:155-71), osteosarcoma, osteomyelitis, bronchiectasis, chronicpulmonary obstructive disease, skin and eye diseases, periodontitis,osteoporosis, rheumatoid arthritis, ulcerative colitis, inflammatorydisorders, tumor growth and invasion (Stetler-Stevenson et al., Annu.Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys.Acta 1987, 907:191-217; Li et al., Mol. Carcinog. 1998, 22:84-89)),metastasis, acute lung injury, stroke, ischemia, diabetes, aortic orvascular aneurysms, skin tissue wounds, dry eye, bone and cartilagedegradation (Greenwald et al., Bone 1998, 22:33-38; Ryan et al., Curr.Op. Rheumatol. 1996, 8; 238-247). Other MMPAS include those described inU.S. Pat. Nos. 5,459,135; 5,321,017; 5,308,839; 5,258,371; 4,935,412;4,704,383, 4,666,897, and RE 34,656, incorporated herein by reference intheir entirety.

In another embodiment, the tetracycline compound responsive state iscancer. Examples of cancers which the tetracycline compounds of theinvention may be useful to treat include all solid tumors, i.e.,carcinomas e.g., adenocarcinomas, and sarcomas. Adenocarcinomas arecarcinomas derived from glandular tissue or in which the tumor cellsform recognizable glandular structures. Sarcomas broadly include tumorswhose cells are embedded in a fibrillar or homogeneous substance likeembryonic connective tissue. Examples of carcinomas which may be treatedusing the methods of the invention include, but are not limited to,carcinomas of the prostate, breast, ovary, testis, lung, colon, andbreast. The methods of the invention are not limited to the treatment ofthese tumor types, but extend to any solid tumor derived from any organsystem. Examples of treatable cancers include, but are not limited to,colon cancer, bladder cancer, breast cancer, melanoma, ovariancarcinoma, prostatic carcinoma, lung cancer, and a variety of othercancers as well. The methods of the invention also cause the inhibitionof cancer growth in adenocarcinomas, such as, for example, those of theprostate, breast, kidney, ovary, testes, and colon.

In an embodiment, the tetracycline responsive state of the invention iscancer. The invention pertains to a method for treating a subjectsuffering or at risk of suffering from cancer, by administering aneffective amount of a substituted tetracycline compound, such thatinhibition cancer cell growth occurs, i.e., cellular proliferation,invasiveness, metastasis, or tumor incidence is decreased, slowed, orstopped. The inhibition may result from inhibition of an inflammatoryprocess, down-regulation of an inflammatory process, some othermechanism, or a combination of mechanisms. Alternatively, thetetracycline compounds may be useful for preventing cancer recurrence,for example, to treat residual cancer following surgical resection orradiation therapy. The tetracycline compounds useful according to theinvention are especially advantageous as they are substantiallynon-toxic compared to other cancer treatments. In a further embodiment,the compounds of the invention are administered in combination withstandard cancer therapy, such as, but not limited to, chemotherapy.

In one embodiment, the tetracycline compounds of the invention areantioxidants. The term “antioxidant” refers to compounds that mayprotect cells from the damage caused by unstable molecules, such as freeradical molecules. Free radical moieties lead to the oxidation of tissueresulting in, for example, aging, cancer, heart disease and the like.Without being bound to any particular theory, antioxidants may preventfree radicals from oxidizing sensitive biological molecules found intissues and/or reduce the formation of free radicals.

Examples of tetracycline responsive states also include neurologicaldisorders which include both neuropsychiatric and neurodegenerativedisorders, but are not limited to, such as Alzheimer's disease,dementias related to Alzheimer's disease (such as Pick's disease),Parkinson's and other Lewy diffuse body diseases, demyelination-relateddisorders, senile dementia, Huntington's disease, Gilles de laTourette's syndrome, multiple sclerosis (e.g., including but not limitedto, relapsing and remitting multiple sclerosis, primary progressivemultiple sclerosis, and secondary progressive multiple sclerosis),amylotrophic lateral sclerosis (ALS), progressive supranuclear palsy,epilepsy, and Creutzfeldt-Jakob disease; autonomic function disorderssuch as hypertension and sleep disorders, and neuropsychiatricdisorders, such as depression, schizophrenia, schizoaffective disorder,Korsakoff's psychosis, mania, anxiety disorders, or phobic disorders;learning or memory disorders, e.g., amnesia or age-related memory loss,attention deficit disorder, dysthymic disorder, major depressivedisorder, mania, obsessive-compulsive disorder, psychoactive substanceuse disorders, anxiety, phobias, panic disorder, as well as bipolaraffective disorder, e.g., severe bipolar affective (mood) disorder(BP-1), bipolar affective neurological disorders, e.g., migraine andobesity. Further neurological disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

In one embodiment, the tetracycline responsive state is a demyelinationrelated disorder. The term “demyelination-related disorders” includesdisorders which are associated with, caused by, or result indemyelination. Demyelination is a major underlying factor responsiblefor the symptoms of multiple sclerosis. Demyelination is the destructiveremoval of myelin, an insulating and protective fatty protein whichsheaths the long extensions of neurons called axons. During relapses ofmultiple sclerosis, patches of white matter in the central nervoussystem that normally contain tracts of myelinated neurons becomeinflamed and lose their myelin. These patches of demyelination are knownas lesions.

Not to be bound by theory, but it is believed that the body's own immunesystem is at least partially responsible. Acquired immune system cells,called T-cells, are known to be present at the site of lesions. Otherimmune system cells called macrophages (and possibly mast cells as well)also contribute to the damage.

Myelin is produced by oligodendrocytes in the central nervous system.One oligodendrocyte produces myelin for several axons and one axon hasseveral oligodendrocytes producing its myelin. In multiple sclerosis,not only is the myelin destroyed but also the oligodendrocytes and eventhe axons themselves.

Axons use an electrochemical mechanism to transmit nerve impulses—theaction potential. This requires sodium and potassium ions to passthrough a semi-permeable membrane around the nerve. It is believed thatthe myelin not only insulates and encases this electrochemical processbut also actively assists it. When axons become demyelinated, theytransmit the nerve impulses 10 times slower than normal myelinatedcells.

The term “demyelination-related disorder” includes multiple sclerosis,central pontine myelinolysis, leukodystrophies, acute disseminatedencephalomyelitis, progressive multifocal leukoencephalopathy, subacutesclerosing panencephalitis, and other disorders caused or characterizedby demyelination of neurons.

The term “multiple sclerosis” includes all forms of multiple sclerosis.It includes relapsing and remitting multiple sclerosis, primaryprogressive multiple sclerosis, and secondary progressive multiplesclerosis.

The language “in combination with” another therapeutic agent ortreatment includes co-administration of the tetracycline compound andwith the other therapeutic agent or treatment, administration of thetetracycline compound first, followed by the other therapeutic agent ortreatment and administration of the other therapeutic agent or treatmentfirst, followed by the tetracycline compound. The other therapeuticagent may be any agent who is known in the art to treat, prevent, orreduce the symptoms of an IPAS and another tetracycline responsivestate. Furthermore, the other therapeutic agent may be any agent ofbenefit to the patient when administered in combination with theadministration of an tetracycline compound. In one embodiment, thecancers treated by methods of the invention include those described inU.S. Pat. Nos. 6,100,248; 5,843,925; 5,837,696; or 5,668,122,incorporated herein by reference in their entirety.

In another embodiment, the tetracycline compound responsive state isdiabetes, e.g., juvenile diabetes, diabetes mellitus, diabetes type I,or diabetes type II. In a further embodiment, protein glycosylation isnot affected by the administration of the tetracycline compounds of theinvention. In another embodiment, the tetracycline compound of theinvention is administered in combination with standard diabetictherapies, such as, but not limited to insulin therapy. In a furtherembodiment, the IPAS includes disorders described in U.S. Pat. Nos.5,929,055; and 5,532,227, incorporated herein by reference in theirentirety.

In another embodiment, the tetracycline compound responsive state is abone mass disorder. Bone mass disorders include disorders where asubjects bones are disorders and states where the formation, repair orremodeling of bone is advantageous.

For examples bone mass disorders include osteoporosis (e.g., a decreasein bone strength and density), bone fractures, bone formation associatedwith surgical procedures (e.g., facial reconstruction), osteogenesisimperfecta (brittle bone disease), hypophosphatasia, Paget's disease,fibrous dysplasia, osteopetrosis, myeloma bone disease, and thedepletion of calcium in bone, such as that which is related to primaryhyperparathyroidism. Bone mass disorders include all states in which theformation, repair or remodeling of bone is advantageous to the subjectas well as all other disorders associated with the bones or skeletalsystem of a subject which can be treated with the tetracycline compoundsof the invention. In a further embodiment, the bone mass disordersinclude those described in U.S. Pat. Nos. 5,459,135; 5,231,017;5,998,390; 5,770,588; RE 34,656; 5,308,839; 4,925,833; 3,304,227; and4,666,897, each of which is hereby incorporated herein by reference inits entirety.

In another embodiment, the tetracycline compound responsive state isacute lung injury. Acute lung injuries include adult respiratorydistress syndrome (ARDS), post-pump syndrome (PPS), and trauma. Traumaincludes any injury to living tissue caused by an extrinsic agent orevent. Examples of trauma include, but are not limited to, crushinjuries, contact with a hard surface, or cutting or other damage to thelungs.

The invention also pertains to a method for treating acute lung injuryby administering a substituted tetracycline compound of the invention.

The tetracycline responsive states of the invention also include chroniclung disorders. The invention pertains to methods for treating chroniclung disorders by administering a tetracycline compound, such as thosedescribed herein. The method includes administering to a subject aneffective amount of a substituted tetracycline compound such that thechronic lung disorder is treated. Examples of chronic lung disordersinclude, but are not limited, to asthma, cystic fibrosis, and emphysema.In a further embodiment, the tetracycline compounds of the inventionused to treat acute and/or chronic lung disorders such as thosedescribed in U.S. Pat. Nos. 5,977,091; 6,043,231; 5,523,297; and5,773,430, each of which is hereby incorporated herein by reference inits entirety.

In yet another embodiment, the tetracycline compound responsive state isischemia, stroke, or ischemic stroke. The invention also pertains to amethod for treating ischemia, stroke, or ischemic stroke byadministering an effective amount of a substituted tetracycline compoundof the invention. In a further embodiment, the tetracycline compounds ofthe invention are used to treat such disorders as described in U.S. Pat.Nos. 6,231,894; 5,773,430; 5,919,775 or 5,789,395, incorporated hereinby reference.

In another embodiment, the tetracycline compound responsive state is askin wound. The invention also pertains, at least in part, to a methodfor improving the healing response of the epithelialized tissue (e.g.,skin, mucusae) to acute traumatic injury (e.g., cut, burn, scrape,etc.). The method may include using a tetracycline compound of theinvention (which may or may not have antibacterial activity) to improvethe capacity of the epithelialized tissue to heal acute wounds. Themethod may increase the rate of collagen accumulation of the healingtissue. The method may also decrease the proteolytic activity in theepthithelialized tissue by decreasing the collagenolytic and/orgellatinolytic activity of MMPs. In a further embodiment, thetetracycline compound of the invention is administered to the surface ofthe skin (e.g., topically). In a further embodiment, the tetracyclinecompound of the invention used to treat a skin wound, and other suchdisorders as described in, for example, U.S. Pat. Nos. 5,827,840;4,704,383; 4,935,412; 5,258,371; 5,308,839, 5,459,135; 5,532,227; and6,015,804; each of which is incorporated herein by reference in itsentirety.

In yet another embodiment, the tetracycline compound responsive state isan aortic or vascular aneurysm in vascular tissue of a subject (e.g., asubject having or at risk of having an aortic or vascular aneurysm,etc.). The tetracycline compound may by effective to reduce the size ofthe vascular aneurysm or it may be administered to the subject prior tothe onset of the vascular aneurysm such that the aneurysm is prevented.In one embodiment, the vascular tissue is an artery, e.g., the aorta,e.g., the abdominal aorta. In a further embodiment, the tetracyclinecompounds of the invention are used to treat disorders described in U.S.Pat. Nos. 6,043,225 and 5,834,449, incorporated herein by reference intheir entirety.

Bacterial infections may be caused by a wide variety of gram positiveand gram negative bacteria. The compounds of the invention may be usefulas antibiotics against organisms which are resistant to othertetracycline compounds. The antibiotic activity of the tetracyclinecompounds of the invention may be determined using the method discussedin Example 2, or by using the in vitro standard broth dilution methoddescribed in Waitz, J. A., National Commission for Clinical LaboratoryStandards, Document M7-A2, vol. 10, no. 8, pp. 13-20, 2^(nd) edition,Villanova, Pa. (1990). In one embodiment, the tetracycline compounds arenon-anti-bacterial (e.g., exhibit an MIC greater than about 8 μg/mL, asdescribed in Example 2).

The tetracycline compounds may also be used to treat infectionstraditionally treated with tetracycline compounds such as, for example,rickettsiae; a number of gram-positive and gram-negative bacteria; andthe agents responsible for lymphogranuloma venereum, inclusionconjunctivitis, psittacosis. The tetracycline compounds may be used totreat infections of, e.g., K. pneumoniae, Salmonella, E. hirae, A.baumanii, B. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E.coli, S. aureus or E. faecalis. In one embodiment, the tetracyclinecompound is used to treat a bacterial infection that is resistant toother tetracycline antibiotic compounds. The tetracycline compound ofthe invention may be administered with a pharmaceutically acceptablecarrier. The tetracycline compounds of the invention may also be used totreat fungal disorders, viral disorders, parasitic disorders, and otherdisorders described in WO 03/005971, WO 02/085303, WO 02/072022, WO02/072031, WO 01/52858, and U.S. Ser. No. 10/692,764, each of which isincorporated herein by reference in its entirety.

In another embodiment, the tetracycline responsive state is a disordertreated by modulation of RNA.

The term “disorders treatable by modulation of RNA” or “DTMR” includesviral, neurodegenerative and other disorders which are caused or relatedto RNA function, structure, amounts and/or other activities of RNA whichare lower or higher than desired and those disorders treatable bycompounds described herein. Examples of DTMR include viral disorders(e.g., retroviral disorders (e.g., HIV, etc.), disorders caused by humanrhinovirus RNA and proteins, VEE virus, Venezuelan equine encephalitisvirus, eastern X disease, West Nile virus, bacterial spot of peach,camelpox virus, potato leafroll virus, stubborn disease and infectiousvariegations of citrus seedlings, viral protein synthesis in Escherichiacoli infected with coliphage MS2, yellow viruses, citrus greeningdisease, ratoon stunting disease, European yellows of plants, inclusionconjunctivitis virus, meningopneumonitis virus, trachoma virus, hogplague virus, ornithosis virus, influenza virus, rabies virus, viralabortion in ungulates, pneumonitis, and cancer.

Other exemplary DTMRs include disorders caused by, or associated withsplicing. For example, some disorders associated with defects inpre-mRNA processing result from a loss of function due to mutations inregulatory elements of a gene. Examples of such mutations are describedin Krawczak et al. (1992) Hum. Genet, 90:41-54; and Nakai et al. (1994)Gene 14:171-177. Other DTMR include disorders which have been attributedto a change in trans-acting factors. Examples of DTMRs which areassociated with splicing include those described in Philips et al.(2000), Cell. Mol. Life Sci., 57:235-249), as well as, FTDP-17(frontotemporal dementia with parkinsonism) and β-thalassemia.

Certain DTMRs associated with splicing include those which are generatedby point mutations that either destroy splice-sites or generate newcryptic sites in the vicinity of normally used exons. Examples of suchDTMRs include cystic fibrosis (Friedman et al. (1999) J. Biol. Chem.274:36193-36199), muscular dystrophy (Wilton et al. (1999) Neuromuscul.Disord. 9:330-338), and eosinophilic diseases (Karras et al., (2000)Mol. Pharamcol. 58:380-387).

Other DTMRs include cancers which may change splicing patterns duringcancer formation and progression. Example of such cancers include, butare not limited to leukemia, colon/rectal cancer, myeloid leukemia,breast cancer, gastric carcinomas, acute leukemia, multiple myeloma,myeloid cell leukemia, lung cancer, prostate cancer, etc. Addition DTMRsassociated with splicing are discussed in Stoss et al., (2000), GeneTher. Mol. Biol. 5:9-30).

Another example of a DTMR is a cancer in which treatment of the cancercells with a tetracycline compound results in the modulation of RNA,where the modulation of RNA increases the susceptibility of the cell toa second agent, e.g., a chemotherapeutic agent. Such DTMRs can betreated using a combination of the tetracycline compound and achemotherapeutic agent. Exemplary cancers include those in which thetetracycline compound modulates the form of BCL expressed by the cells.

Other DTMRs include disorders wherein particular ribozymes are presentin aberrant quantities. Examples include breast cancer, hepatitis Cvirus (HCV), liver cirrhosis, and heptacellular carcinoma.

The language “effective amount” of the compound is that amount necessaryor sufficient to treat or prevent a tetracycline compound responsivestate. The effective amount can vary depending on such factors as thesize and weight of the subject, the type of illness, or the particulartetracycline compound. For example, the choice of the tetracyclinecompound can affect what constitutes an “effective amount”. One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thetetracycline compound without undue experimentation.

The invention also pertains to methods of treatment againstmicroorganism infections and associated diseases. The methods includeadministration of an effective amount of one or more tetracyclinecompounds to a subject. The subject can be either a plant or,advantageously, an animal, e.g., a mammal, e.g., a human.

In the therapeutic methods of the invention, one or more tetracyclinecompounds of the invention may be administered alone to a subject, ormore typically a compound of the invention will be administered as partof a pharmaceutical composition in mixture with conventional excipient,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, oral or other desired administrationand which do not deleteriously react with the active compounds and arenot deleterious to the recipient thereof.

The invention also pertains to pharmaceutical compositions comprising atherapeutically effective amount of a tetracycline compound (e.g., atetracycline compound of the formula I, II, or otherwise describedherein) and, optionally, a pharmaceutically acceptable carrier.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allow both to perform their intended function, e.g., treat orprevent a tetracycline responsive state. Suitable pharmaceuticallyacceptable carriers include but are not limited to water, saltsolutions, alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds of the invention.

The tetracycline compounds of the invention that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of the tetracycline compounds of theinvention that are basic in nature are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)]salts.Although such salts must be pharmaceutically acceptable foradministration to a subject, e.g., a mammal, it is often desirable inpractice to initially isolate a tetracycline compound of the inventionfrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent and subsequently convert the latterfree base to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained. The preparation of other tetracycline compounds of theinvention not specifically described in the foregoing experimentalsection can be accomplished using combinations of the reactionsdescribed above that will be apparent to those skilled in the art.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention that are acidic in natureare capable of forming a wide variety of base salts. The chemical basesthat may be used as reagents to prepare pharmaceutically acceptable basesalts of those tetracycline compounds of the invention that are acidicin nature are those that form non-toxic base salts with such compounds.Such non-toxic base salts include, but are not limited to those derivedfrom such pharmaceutically acceptable cations such as alkali metalcations (e.g., potassium and sodium) and alkaline earth metal cations(e.g., calcium and magnesium), ammonium or water-soluble amine additionsalts such as N-methylglucamine-(meglumine), and the loweralkanolammonium and other base salts of pharmaceutically acceptableorganic amines. The pharmaceutically acceptable base addition salts oftetracycline compounds of the invention that are acidic in nature may beformed with pharmaceutically acceptable cations by conventional methods.Thus, these salts may be readily prepared by treating the tetracyclinecompound of the invention with an aqueous solution of the desiredpharmaceutically acceptable cation and evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively, alower alkyl alcohol solution of the tetracycline compound of theinvention may be mixed with an alkoxide of the desired metal and thesolution subsequently evaporated to dryness.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention and pharmaceuticallyacceptable salts thereof can be administered via either the oral,parenteral or topical routes. In general, these compounds are mostdesirably administered in effective dosages, depending upon the weightand condition of the subject being treated and the particular route ofadministration chosen. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

The pharmaceutical compositions of the invention may be administeredalone or in combination with other known compositions for treatingtetracycline responsive states in a subject, e.g., a mammal. Preferredmammals include pets (e.g., cats, dogs, ferrets, etc.), farm animals(cows, sheep, pigs, horses, goats, etc.), lab animals (rats, mice,monkeys, etc.), and primates (chimpanzees, humans, gorillas). Thelanguage “in combination with” a known composition is intended toinclude simultaneous administration of the composition of the inventionand the known composition, administration of the composition of theinvention first, followed by the known composition and administration ofthe known composition first, followed by the composition of theinvention. Any of the therapeutically composition known in the art fortreating tetracycline responsive states can be used in the methods ofthe invention.

The tetracycline compounds of the invention may be administered alone orin combination with pharmaceutically acceptable carriers or diluents byany of the routes previously mentioned, and the administration may becarried out in single or multiple doses. For example, the noveltherapeutic agents of this invention can be administered advantageouslyin a wide variety of different dosage forms, i.e., they may be combinedwith various pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, aqueous suspensions, injectable solutions, elixirs, syrups,and the like. Such carriers include solid diluents or fillers, sterileaqueous media and various non-toxic organic solvents, etc. Moreover,oral pharmaceutical compositions can be suitably sweetened and/orflavored. In general, the therapeutically-effective compounds of thisinvention are present in such dosage forms at concentration levelsranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

For parenteral administration (including intraperitoneal, subcutaneous,intravenous, intradermal or intramuscular injection), solutions of atherapeutic compound of the present invention in either sesame or peanutoil or in aqueous propylene glycol may be employed. The aqueoussolutions should be suitably buffered (preferably pH greater than 8) ifnecessary and the liquid diluent first rendered isotonic. These aqueoussolutions are suitable for intravenous injection purposes. The oilysolutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art. Forparenteral application, examples of suitable preparations includesolutions, preferably oily or aqueous solutions as well as suspensions,emulsions, or implants, including suppositories. Therapeutic compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

Additionally, it is also possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin. Examples of methods of topical administration include transdermal,buccal or sublingual application. For topical applications, therapeuticcompounds can be suitably admixed in a pharmacologically inert topicalcarrier such as a gel, an ointment, a lotion or a cream. Such topicalcarriers include water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils. Otherpossible topical carriers are liquid petrolatum, isopropylpalmitate,polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% inwater, sodium lauryl sulfate 5% in water, and the like. In addition,materials such as anti-oxidants, humectants, viscosity stabilizers andthe like also may be added if desired.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is derivatized withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods ofthe invention also will have significant veterinary applications, e.g.for treatment of livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys and the like;horses; and pets such as dogs and cats. Also, the compounds of theinvention may be used to treat non-animal subjects, such as plants.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

In general, compounds of the invention for treatment can be administeredto a subject in dosages used in prior tetracycline therapies. See, forexample, the Physicians' Desk Reference. For example, a suitableeffective dose of one or more compounds of the invention will be in therange of from 0.01 to 100 milligrams per kilogram of body weight ofrecipient per day, preferably in the range of from 0.1 to 50 milligramsper kilogram body weight of recipient per day, more preferably in therange of 1 to 20 milligrams per kilogram body weight of recipient perday. The desired dose is suitably administered once daily, or severalsub-doses, e.g. 2 to 5 sub-doses, are administered at appropriateintervals through the day, or other appropriate schedule. It will alsobe understood that normal, conventionally known precautions will betaken regarding the administration of tetracyclines generally to ensuretheir efficacy under normal use circumstances. Especially when employedfor therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

Furthermore, the invention also pertains to the use of a tetracyclinecompound of formula I, II, or a compound otherwise described herein forthe preparation of a medicament. The medicament may include apharmaceutically acceptable carrier and the tetracycline compound is aneffective amount, e.g., an effective amount to treat a tetracyclineresponsive state.

EXEMPLIFICATION OF THE INVENTION

Compounds of the invention may be made as described below and/or byusing literature techniques known to those of ordinary skill in the art.

Example 1 Synthesis of 12-Dehydrodoxycycline

Triethylamine was added to a solution of doxycycline (1 g, 2.2 mmole) in15 ml of methanol to bring the pH to about 9. Then, 426 mg of sodiumborohydride (5 eq) was added to this mixture portionwise. The resultingreaction mixture was stirred at room temperature for several hours. Thereaction was monitored by analytical HPLC and LCMS [MS: 445(for startingmaterial) and MS 447(for product)]. The solvent was removed and theresidue was diluted with water. The aqueous solution was then extractedwith n-butanol (2×). The combined organic fractions were evaporatedunder reduced pressure to give the alcohol. This material wasredissolved in 20 ml of trifluoroacetic acid and heated at 60 forseveral hours. The reaction was monitored by analytical HPLC and LCMS[MS: 447 for the alcohol and 429 for the dehydrated material). At thecompletion of the reaction, the TFA was evaporated and the residue wasdissolved in a mixture of methanol/water (3:1). The solution wasfiltered and the desired material isolated via preparative HPLC. About250 mg of light yellow solid was obtained (MS: 429). The chemicalstructure was further characterized by NMR.

1 mmol of 12-dehydrodoxycycline trifluoroacetate in 15 ml of DMF wasreacted with 4 equivalent of amine and in the presence of 1 eq. ofInCl₃. The reaction mixture was stirred at room temperature for severalhours. The desired material was isolated via preparative HPLC.

12-Dehydrodoxycycline

(4S,4aR,5S,5aR,6R,12aR)-4-Dimethylamino-3,5,10,12a-tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (MS (M+H): 429.4)

The ¹H, ¹³C chemical shifts, and ³J_(H-H) coupling constants of12-dehydrodoxycycline^(a) are shown in Table 2 below.

TABLE 2 Position ¹³C δ^(b) Δ^(c) ¹H δ^(b) Δ^(c) ³J_(H-H) ^(d,e)  1  1197.08 1.43 — — — —  2  2 97.00 0.81 — — — —  2a 2a-CONH₂ 174.57 0.422a-CONHa N/O — N/O — — — — 2a-CONHb N/O — N/O  3  3 187.96 −0.25 3-OHN/O — N/O  4  4 67.75 0.60  4β 4.43 −0.02 d, 0.9 4-NMe₂-a 4-NMe₂-a 43.810.30 4-NMe₂-a 2.99 −0.01 s 4-NMe₂-b 4-NMe₂-b 42.52 0.49 4-NMe₂-b 2.980.05 s  4a 4a 44.33 1.19 4a 2.69 −0.14 dd, 11.4, 1.2  5  5 71.56 1.515-OH N/O — N/O — — — —  5β 3.61 0.04 dd, 7.8, 7.8  5a  5a 50.61 2.73  5a2.63 0.04 m  6  6 40.25 0.09 6-Me 1.60 0.05 d, 6.6 — 6-Me 17.68 1.40  6β2.81 0.06 m  6a  6a 149.68 0.44 — — — —  7  7 117.22 0.52  7 6.96 0.01d, 7.8  8  8 138.33 0.45  8 7.48 −0.01 at, 7.8, 8.4  9  9 117.00 −0.23 9 6.84 0.00 d, 8.1 10 10 164.74 1.18 10-OH N/O — N/O 10a 10a 117.370.19 — — — — 11 11 192.57 −1.41 — — — — 11a 11a 138.38 29.82 — — — — 1212 136.78 −35.66 12 7.11 — d, 2.1 12a 12a 72.55 −2.06 12a-OH N/O — N/O^(a)Chemical shifts in methanol-d₄. “N/O” means “Not Observed”.^(b)Chemical shifts are in ppm, referenced to TMS as internal standardat 0 ppm. ^(c)Differences are calculated by subtracting chemical shiftsfor Doxycycline from their corresponding chemical shifts in12-dehydrodoxycycline. ^(d)Coupling constants are in Hertz. ^(e)“a”,“b”, “s”, “d”, “t”, and “q” mean “apparent”, “broad”, “singlet”,“doublet”, “triplet”, and “quartet” respectively.

Other compounds synthesized using similar methods include:

9-[(2,2-Dimethyl-propylamino)-methyl]-12-dehydrodoxycline

(4S,4aR,5S,5aR,6R,12aR)-4-Dimethylamino-9-[(2,2-dimethyl-propylamino)-methyl]-3,5,10,12a-tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (MS (M+H): 528.6)

9-Amino-12-dehydrodoxycline

(4S,4aR,5S,5aR,6R,12aR)-9-Amino-4-dimethylamino-3,5,10,12a-tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (MS (M+H):444.4)

9-bis-Dimethylamino-12-dehydrodoxycline

(4S,4aR,5S,5aR,6R,12aR)-4,9-Bis-dimethylamino-3,5,10,12a-tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (MS (M+H): 472.5)

12-Butylamino-12-dehydrodoxycycline

(4S,4aR,5S,5aR,6R,12aS)-12-Butylamino-4-dimethylamino-3,5,10,12a-tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,11a,12,12a-decahydro-naphthacene-2-carboxylic acid amide (MS:502.5)

12-Methylamino-12-dehydrodoxycycline

(4S,4aR,5S,5aR,6R,12aS)-4-Dimethylamino-3,5,10,12a-tetrahydroxy-6-methyl-12-methylamino-1,11-dioxo-1,4,4a,5,5a,6,11,11a,12,12a-decahydro-naphthacene-2-carboxylicacid amide (MS: 460.2)

12-(2,2-dimethyl-propylamino)-12-dehydrodoxycycline

(4S,4aR,5S,5aR,6R,12aS)-4-Dimethylamino-12-(2,2-dimethyl-propylamino)-3,5,10,12a-tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,11a,12,12a-decahydro-naphthacene-2-carboxylicacid amide (MS: 516.6)

(4S,4aR,5S,5aR,6R,12aS)-4-Dimethylamino-3,5,10,12a-tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,11a,12,12a-decahydro-naphthacene-2-carboxylicacid amide (MS: 431).

12-dehydrominocycline

(4S,4aS,5aR,12aR)-4,7-Bis-dimethylamino-3,10,12a-trihydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (MS:M+H 442.5).

Example 2 In vitro Minimum Inhibitory Concentration (MIC) Assay

The following assay was used to determine the efficacy of tetracyclinecompounds against common bacteria. 2 mg of each compound were dissolvedin 100 μl of DMSO. The solution was then added to cation-adjustedMueller Hinton broth (CAMHB), which resulted in a final compoundconcentration of 200 g per ml. The tetracycline compound solutions werediluted to 50 μL volumes, with a test compound concentration of 0.098μg/ml. Optical density (OD) determinations were made from freshlog-phase broth cultures of the test strains. Dilutions were made toachieve a final cell density of 1×10⁶ CFU/ml. At OD=1, cell densitiesfor different genera were approximately:

E. coli 1 × 10⁹ CFU/ml S. aureus 5 × 10⁸ CFU/ml Enterococcus sp. 2.5 ×10⁹ CFU/ml 

50 μl of the cell suspensions were added to each well of microtiterplates. The final cell density should be approximately 5×10⁵ CFU/ml.These plates are incubated at 35° C. in an ambient air incubator forapproximately 18 hours. The plates are read with a microplate reader andare visually inspected when necessary. The MIC was defined as the lowestconcentration of the tetracycline compound that inhibits growth.

Compounds which were found to have low antibacterial activity include12-dehydrodoxycycline, 9-bis-dimethylamino-12-dehydrodoxycline and9-Amino-12-dehydrodoxycline.

Example 3 Demyelination-Related Disease Animal Model (DRDAM): In vivoExperimental Autoimmune Encephalomyelitis (EAE) Murine Model

In this example, a mouse model was used to determine the ability of thetetracycline compounds to treat demyelination related diseases. Othermodels which can be used are described in Brundula V. et al. Brain 2002June; 125(Pt 6):1297-308 and Popovic N. et al. Ann Neurol. 2002February; 51(2):215-23.

To induce EAE, 6 week old C57BL/6 female mice were injectedsubcutaneously with 200 ug of myelin oligodendrocyte glycoproteinpeptide (MOG 35-55) emulsified in 200 μL of complete Freund's adjuvantcontaining an additional 4 mg/mL of heat killed M. tuberculosis. Micewere additionally injected intravenously (tail vein) with 150 ng oflyophilized pertussis toxin resuspended in 150 μL PBS at time 0 andagain 48 hours later. In this model, the mice developed progressiveposterior to anterior advancing paralysis with symptoms first appearingat 10-12 days and progressing to severe paralysis by approximately 21days. Severity of EAE was scored daily according to the followingcriteria: 0, healthy; 1, limp tail; 2, partial paralysis of one or twohind limbs; 3, complete paralysis of the hind limbs; 4, complete hindlimb paralysis and forelimb paraparesis; 5, moribund. All testexperiments included control groups of EAE induced mice treated withvehicle only or treated with minocycline, with 10 mice per group.Treatment began on day 10 (onset of disease) as IP injections with 50mg/kg of minocycline, test tetracycline compound or vehicle alone eachday until the end of the study. 12-Dehydrodoxycycline and12-methylamino-12-dehydrodoxycycline were found to have activity similarto or better than minocycline.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

1. A compound selected from the group consisting of:

and pharmaceutically acceptable salts, esters, prodrugs and enantiomersthereof.
 2. A pharmaceutical composition comprising a compound of claim1 and a pharmaceutically acceptable carrier.
 3. The pharmaceuticalcomposition of claim 2, wherein said compound is present in an amounteffective to ameliorate a disease selected from: bacterial infection,multiple sclerosis, central pontine myelinolysis, leukodystrophy, acutedisseminated encephalomyelitis, progressive multifocalleukoencephalopathy, and subacute sclerosing panencephalitis.
 4. Amethod for ameliorating a disease selected from: bacterial infection,multiple sclerosis, central pontine myelinolysis, leukodystrophy, acutedisseminated encephalomyelitis, progressive multifocalleukoencephalopathy, and subacute sclerosing panencephalitis in asubject in need thereof, comprising administering to said subject aneffective amount of a compound of claim 1, such that said disease isameliorated.
 5. The method of claim 4, wherein said subject is a mammal.6. The method of claim 4, wherein said subject is a human.
 7. The methodof claim 4, wherein said disease is bacterial infection.
 8. The methodof claim 4, wherein said disease is central pontine myelinolysis.
 9. Themethod of claim 4, wherein said disease is leukodystrophy.
 10. Themethod of claim 4, wherein said disease is acute disseminatedencephalomyelitis.
 11. The method of claim 4, wherein said disease isprogressive multifocal leukoencephalopathy.
 12. The method of claim 4,wherein said disease is subacute sclerosing panencephalitis.
 13. Themethod of claim 4, wherein said disease is multiple sclerosis.
 14. Themethod of claim 13, wherein said multiple sclerosis is relapsing orremitting multiple sclerosis, primary progressive multiple sclerosis, orsecondary progressive multiple sclerosis.
 15. The tetracycline compoundof claim 1, wherein the tetracycline compound is an anti-oxidant.